Detection by Immunoassays
HP Dwivedi and G Devulder, bioMerieux, Inc., Hazelwood, MO, USA
VK Juneja, Eastern Regional Research Center, USDA-Agricultural Research Service, Wyndmoor, PA, USA
Ó 2014 Elsevier Ltd. All rights reserved.
Antigenic Makeup of Salmonella
Salmonella serovars exhibit three different cell-surface anti-
gens. The somatic (O) antigens, which are present on the
outer membrane, are part of lipopolysaccharides (LPS)
moieties. The heat-stable somatic antigens of Salmonella
include both specific determinants and/or nondiscriminatory
antigens. The Salmonella subspecies are divided into over 50
serogroups based on the presence of somatic (O) antigens,
including A, B, C1, C2, D1, E1, E2, E3, and E4. Although
specific somatic antigens (LPS) have considerable discrimi-
natory power but cross-reaction can occur between the
common antigens among the different serotypes of Salmo-
nella. The anti-Salmonella antibodies could cross-react with the
antigenically closely related species of certain genera. Gene
clusters controlling the somatic antigen of Salmonella that has
closer similarity with the somatic antigen of other genera are
supposed to have originated from a common ancestor. The
O-antigen modifications could possibly be attributed to the
induction by prophage genes outside the gene cluster during
the evolutionary process of species divergence.
The flagellar (H) antigens, which are heat-labile proteins, are
associated with peritrichous flagella. Variation of the flagellar
antigens between two forms (biphasic antigenic variation), H1
and H2, in which the flagellar subunit consists of FliC or
FliB protein, respectively, is noticed in serotypes such as
S. Typhimurium. Phase variation is also common among
Salmonella serotypes, and variable expression of flagellin
proteins, resulting in the assembly of flagella with different
structures, can be observed. In the case of biphasic organism, the
phase inversion technique could be used to determine both
phases. The capsular or virulence (Vi) antigen is also available to
screen the Salmonella serotypes (group D). Salmonella serotypes
such as Typhi, Paratyphi C, and Dublin exhibit the capsular
antigens (Vi), which are referred to as K antigens in other
enterobacteriaceae. Serotyping employing Salmonella antigens
could be used for the differentiation among serotypes based on
somatic, flagellar, and capsular antigens using slide agglutina-
tion or tube agglutination tests.
The expression of Salmonella antigens could be affected
by the components of the growth medium. For example,
Salmonella enterica grown on solid medium containing iron,
thiosulfate, hexoses, and amino acids is reported to undergo
cell-surface differentiation such as increased flagellation and
conversion from rough to smooth LPS. Similarly, peptone
constituents of culture medium could induce morphological
differences in S. Typhimurium, consequently affecting sero-
logical identification. Transfer of aflagellate Salmonella from
nutritionally poor media deprived of optimum amounts
of tyrosine into a rich nutrient broth could allow flagella
synthesis, indicating that the aflagellate form is still able
to produce flagella as reported in the study by Gray et al.
(2006).
Encyclopedia of Food Microbiology, Volume 3 http://dx.doi.org/10.1016/B978-0-12-384730-0.00299-8
Enzyme-Linked Immunosorbent Assay
Enzyme-Linked Immunosorbent Assay (ELISA) tests are
applied for the detection of Salmonella antigens or antibodies
in a sample. However, most of the food Salmonella ELISAs are
based on the detection of Salmonella antigens using anti-
Salmonella polyclonal or monoclonal antibodies as primary
antibodies tethered to a solid support for the capture of
antigens. Nonspecific immobilization of the Salmonella anti-
gens in food could also be performed using passive adsorp-
tion on solid surfaces such as polystyrene microtiter plates.
The primary antibodies bind specifically with the Salmonella
antigens if present in food samples, resulting in specific
antigen–antibody complexes. The detection of antigen–
antibody complexes could be performed using secondary
antibodies conjugated to an enzyme (such as alkaline phos-
phatase) in a sandwich format assay. The positive detection is
facilitated by the development of a detectable color due to
enzymatic reaction by conjugate, when an appropriate
substrate (such as p-nitrophenyl phosphate) is applied to the
conjugate. Many other combinations of conjugates and
substrates could also be used such as Horse Radish Peroxidase
conjugate and 3,30 ,5,50 -tetramethylbenzidine substrate.
Washing steps are frequently performed to remove unbound
antibodies and avoid any unspecified bindings to ensure
specificity of the assay. Relative quantification of Salmonella
antigens in unknown samples could be performed by
comparing against the standard curve values to give a positive
or negative call to a sample. ELISAs could be performed in
various formats (such as direct and indirect) other than
sandwich, which is described here.
The direct application of some food samples on solid assay
surfaces may result in nonspecific binding of matrix compo-
nents, which in turn may interfere with antigen–antibody
reactions or lead to nonspecific results. Several ELISAs have
been developed to detect antigens of Salmonella spp. in foods.
Besides primary antibodies used in the capture and immobi-
lization of Salmonella antigens present in food samples, alter-
nate ligands have been employed in enzyme immunoassay for
the detection of Salmonella. For example, polymyxin-ELISA was
reported for the detection of group D salmonellae (including S.
Enteritidis), using polymyxin immobilized in the wells of
microtiter plate as a high-affinity adsorbent for LPS antigens.
Although the sensitivity of ELISAs depends on the food
matrices being analyzed (among other factors), the typical limit
of detection varies from as low as 104 to >105 CFU ml1.
Sensitivity of the assay could further be affected by interference
due to background flora in food matrices, expression of
Salmonella antigens in the food, and growth rate of Salmonella
strains. Specificity of ELISAs for Salmonella detection mostly
relies on the specificity of the antibodies employed. Mono-
clonal antibodies could be more specific, but achieving inclu-
sivity for more than 2400 Salmonella serotypes could be
339
340 SALMONELLA j Detection by Immunoassays

challenging. Similarly, polyclonal antibodies could be more
inclusive, but there could be concern of cross-reactivity with an
antigenically closer genus such as Citrobacter spp.
The enrichment of food samples is required to achieve the
limit of detection by enzyme-based immunoassays. For appli-
cation in several immunoassays, heating of enriched samples is
performed to release the antigens from bacteria attached to the
food matrix. Many modified versions of ELISAs have been
reported that require relatively smaller sample volumes and have
improved detection limits with fewer reports of false-positive
results. Further modifications have been reported to enhance the
sensitivity and multiplexing ability of the assay and to make it
more quantitative. In this effort, fluorogenic and electro-
chemiluminescent reporters have been employed in place of
traditional chromogenic reporter substrates. Automated ELISA
formats are amenable to performing large numbers of sample
analyses with relative ease and rapidity. The choice of conjugate
system also plays an important role in the application of ELISA-
based detection in food matrices. For example, when using
peroxidase conjugates, care should be taken as many food
pathogens express intracellular peroxidases or catalases or both,
thus resulting in nonspecific immune reaction. Alternatively,
conjugates based on alkaline phosphate could be used. Salmo-
nella concentration using immunoconcentration approaches
could be performed upfront of enzyme immunoassay. These
combined immunoconcentration-ELISA approaches could help
reduce the time to detection and achieve enhanced detection
sensitivity. The automated immunoconcentration platforms are
commercially available, which can process several samples in
a single run, thus improving the overall efficiency.
In general, ELISAs are simple, easy to perform, scalable,
and adaptable assays. Some of the commercial ELISAs provide
simplicity of visual detection of color changes due to positive
enzymatic reactions; however, many others are automated.
Commercially available ELISA and immunoconcentration
assays for Salmonella detection in foods are listed in Table 1.
Enzyme-Linked Fluorescent Assay
Enzyme-Linked Fluorescent Assays (ELFAs) are based on
enzymatic reactions similar to ELISA but instead use fluoro-
genic substrates such as 4-methylumbelliferyl phosphate
(4MUP) as the reporter. 4MUP can detect much lower levels
of alkaline phosphatase (109 M) by converting 4MUP into
the fluorescent product 4-methylumbelliferone. However,
colorimetric substrates such as p-nitrophenyl phosphate
(PNP) can detect only 105 M of alkaline phosphatase as it
converts PNP into the yellow pigmented p-nitrophenol. ELFA
is reported to be more sensitive and faster than ELISA. The
automation, rapidity, and ease of use of the automated ELFA
formats have further increased ELFA’s popularity. Application
of ELFA-based assays for the detection of Salmonella in various
food matrices has been widely reported. Alternate ligands
such as aptamers, recombinant phage proteins, and peptides
could also be applied in conjunction with antibodies to
enhance the sensitivities and specificities of ELFA. It must be
noted that the sensitivity of ELFA still relies on the upfront
enrichment of samples using broth media. Commercially
available ELFAs for the detection of Salmonella in foods are
listed in Table 1.
It must be noted that all samples analyzed using enzyme
immunoassays must be confirmed using culture-based proce-
dures to conclude the test results. Until cultural confirmation
procedures are completed, a positive immunoassay should be
considered only a presumptive result. Immunoassays for
detection are not to be confused with definitive serological
methods, such as serotyping, which can be confirmatory.

Table 1 Selected immunology based commercial products for detection and identification of Salmonella in food

Assay name and source Technique Target organism(s)

TECRA Salmonella VIA (3 M) ELISA Salmonella spp.

TECRA Salmonella ULTIMA (3 M) ELISA Salmonella spp.
VIP Goldä for Salmonella (BioControl Systems) Lateral flow immunoassay Salmonella spp.
Revealâ test systems Salmonella (Neogen Corp.) Lateral flow immunoassay Salmonella spp.
Reveal S. Enteritidis (Neogen) Lateral flow immunoassay Salmonella Enteritidis
Oxoid Salmonella Latex Test (Oxoid, Thermo Fisher Latex agglutination Salmonella spp.
Scientific Inc.)
Assurance EIA Salmonella (BioControl Systems) Enzyme immunoassay Salmonella spp.
RapidCheck Salmonella spp. (SDIX) Lateral flow immunoassay Salmonella spp.
RapidCheck Salmonella Enteritidis (SDIX) Lateral flow immunoassay Salmonella Enteritidis
Dynabeadsâ Anti-Salmonella antibody (Invitrogen, Life Immuno-magnetic beads Salmonella
Technologies)
BeadRetrieverä system (Invitrogen, Life Technologies) Immuno-magnetic separation (IMS) system Salmonella and other foodborne pathogens
Pathatrix (Matrix MicroScience, Life Technologies) IMS system Salmonella and other foodborne pathogens
Bioline Salmonella Rapid Test Kit Methods (Bioline) ELISA Salmonella spp.
Microgen Salmonella Latex Kit (KeyDiagnotics) Latex slide agglutination test Salmonella spp.
Wellcolexâ Color Salmonella (Remel, Thermo Fisher Latex agglutination test (detection and Salmonella spp.
Scientific Inc.) serogrouping)
VIDASâ SLM (bioMerieux SA) ELFA Salmonella spp.
VIDASâ ICS (bioMerieux SA) Immunoconcentration Salmonella spp.
VIDASâ UP Salmonella (bioMerieux SA) ELFAa Salmonella spp.
a
Combine specific phage protein technology.

Lateral Flow Immunoassay (LIA)
The Salmonella antigens present in food samples can be visu-
alized using specific antibody coated test strips. The test strip
for lateral flow assay has Salmonella-specific capture antibodies
impregnated in a solid support such as a nitrocellulose
membrane at a defined distance from the sample application
slot. The detection antibodies coupled to colloidal latex or gold
particles are placed near the sample application slot. When an
enriched food sample is applied, the Salmonella antigens bind
with the detection antibody, and the complex moves laterally
toward the impregnated capture antibody due to capillary
action. The antigenic complex and detection antibody in the
moving fluid segregates into two different capture zones, one
specific for the Salmonella antigen–antibody complex and the
other specific for the unbound detection antibody. A positive
test result is visually evident (usually by two colored lines)
when a Salmonella-specific reaction occurs. This differs from the
visible signal caused by the detection of an antibody-only
control (such as a single line). Detection using these strips is
rapid and takes around 5-10 min. Lateral flow immunoassays
have been reported for the detection of S. enterica from a variety
of food matrices. Several lateral flow immunoassays for the
detection of foodborne Salmonella are commercially available
(Table 1). Lateral flow assays are usually reported to have
a high limit of detection; thus sample enrichment prior to test is
required. There are reports of a relatively higher number of
false-positive results using LIA as compared to more traditional
microtiter plate ELISA methods. Serotype-specific LIA for the
detection of S. Enteritidis in poultry products such as eggs are
also commercially available. When applying such assays, it is
important to differentiate S. Enteritidis from closely related
serotypes of Salmonella such as non-Enteritidis group D1
Salmonella (such as S. Berta or S. Dublin). Further, many viru-
lent phage types of S. Enteritidis have been reported; thus the
inclusivity of these in the S. Enteritidis-specific assay becomes
important. Advancements such as automated readers could
further improve the usefulness of this assay format. Overall, the
ease and rapidity of using LIA makes it convenient to perform
preliminary screening of pathogen contamination in foods and
environmental samples, despite its comparatively lower speci-
ficity and sensitivity.
Latex Agglutination Assay
Latex agglutination (LA) is an immunoassay that is performed
mostly for the primary culture screening of Salmonella by mixing
isolated colony from plate with sensitized latex beads linked to
antibodies specific for Salmonella antigens. Visible clumping
indicative of a positive reaction can be compared with positive
and negative control samples. Attention must be paid to observe
the isolates that autoagglutinate; otherwise they can interfere
with interpretation of the result. LA assays are rapid and easy to
perform, providing results within a few minutes. These assays are
mainly used for the presumptive confirmation of the isolated
colonies and could help reduce samples for further confirma-
tion. However, LA can also be performed for detection of
Salmonella directly from enriched food samples. Specificity of LA
assays relies on the specificity of antibodies incorporated in the
SALMONELLA j Detection by Immunoassays 341
assay. Many latex agglutination kits, including Salmonella
serogroups specific tests, are commercially available for the
detection of foodborne pathogens (Table 1).
See also: Salmonella: Introduction; Salmonella: Detection by
Classical Cultural Techniques.
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